To elucidate the role of gemistocytes in astrocytoma progression we assessed the fraction of neoplastic gemistocytes, bcl-2 expression, p 53 mutations, p53 immunoreactivity (Pab 1801), and proliferating activity (MIB-1) in 40 low-grade astrocytomas with histologically proven progression to anaplastic astrocytomas (WHO grade III) or glioblastoma (WHO grade IV). All 11 astrocytomas with more than 5 % of gemistocytes contained a p 53 mutation, whereas the incidence of p 53 mutation in astrocytomas with less than 5 % of gemistocytes was 61% (p=0.017). In low grade astrocytomas, the p53 labeling index (LI) of gernistocytes (7.4%) was significantly higher than in all tumor cells (3.2%, p=O.0014). Gemistocytes showed a significantly higher bcl-2 expression than all tumor cells, with a mean bcl-2 LI of 15.6% versus 2.7% in low grade astrocytomas (p=0.0002). In contrast, gemistocytes showed a significantly lower proliferating activity than the mean of all tumor cells, with 0.5% versus 2.6% (p<0.0001).We analyzed 28 gemistocytic astrocytoma (mean fraction of gemistocytes 35.0±9.9%) for mutation study in the p 53 and PTEN (MMAC1) tumor suppressor genes. Single strand conformation polymorphism (SSCP), followed by direct DNA sequencing of p53 exon 5-8 revealed a mutation in 23 of 28 (82%) of cases. In contrast, non of 15 gemistocytic astrocytomas (WHO grade II) and only two of 11 (18%) of anaplastic gemistocytic astrocytomas (WHO grade III) contained PTEN mutations.These results indicate that the vast majority of gemistocytes are, however, in a non-proliferative state (GO phase of cell cycle), which suggests terminal differentiation. Their accumulation within astrocytomas may be due to bcl-2- mediated escape from apoptosis. P 53 mutations are a genetic hallmark of gemistocytic astrocytomas, whilst PTEN mutations are absent in low-grade and rare in anaplastic gemistocytic astrocytomas.